Callus induction and growth from bulb scale explants of C. asiaticum. (A) Callus initiation after 1 week of inoculation. (B) Callus proliferation after 3 weeks. (Bars = 0.5 cm.)
Callus biomass growth (in grams) of C. asiaticum on (A) 2,4-D alone and (B) α-naphthalene acetic acid (NAA) + 6-benzylamino purine (BAP) (in combination) in amended Murashige and Skoog medium. Each value represents the mean ± standard error of three repeated experiments. Mean values followed by the different superscripts in a same column are significantly different from each other according to Duncan’s multiple range test at P ≤ 0.05 level.
Gas chromatography-mass spectrometry chromatograms of methanolic extracts of (A) bulb scale and (B) bulb scale callus of C. asiaticum representing the retention times with the corresponding peaks. The X-axis represents the time of elution of each compound and the Y-axis represents the intensity of the signal measured by the ion detector.
Venn diagram representing the proportion of phytocompounds detected by gas chromatography-mass spectrometry in bulb scale and callus extracts of C. asiaticum.
Chemical structure of some important phytocompounds identified in both the extracts (in vivo and callus samples). Images were taken from the PubChem compound database, National Centre for Biotechnology Information.
Antioxidant activities (A) 2, 2-diphenyl-1-picrylhydrazyl (DPPH) activity, (B) peroxidase (POD) activity, and (C) superoxide dismutase (SOD) activity of bulb scale and bulb scale callus of C. asiaticum. Each value represents the mean ± standard error of three replicates. *Significant at P ≤ 0.05 according to Student’s unpaired t test.
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Crinum asiaticum is an important medicinal and ornamental plant known for its anticancer, analgesic, antiviral, anti-inflammatory, and antifungal properties attributable to its diverse classes of compounds. However, the plant is being overexploited in its native habitat because of its therapeutically significant phytocompounds, leading to a decline in its population. Thus, this study aimed to establish a callus culture system and detect and compare the metabolites content in both in vivo and in vitro regenerated calli. In vitro callus culture of C. asiaticum was initiated from bulb scales on Murashige and Skoog medium supplemented with different concentrations of plant growth regulators. Afterward, gas chromatography-mass spectrometry (GC-MS) analyses of methanol extracts of bulb scale and bulb scale-derived calli were performed. The antioxidant potentials of both the samples were evaluated by determining the 2, 2-diphenyl-1-picrylhydrazyl (DPPH), peroxidase (POD), and superoxide dismutase (SOD) activities. The medium fortified with 0.5 mg/L 2,4-dichlorophenoxyacetic acid (2, 4-D) produced profuse callus with a frequency of 95.83%. The GC-MS analysis of both extracts confirmed the presence of 34 and 31 bioactive compounds, respectively. Various phytochemicals such as gamma-sitosterol, squalene, vitamin E, linolenic acid, and guanosine were present in both extracts. The results of all the anti-oxidant assays (DPPH, POD, and SOD) revealed higher antioxidant activities in callus compared with those in bulb scale tissue. This study proved the potential of using callus culture on a larger scale as a source of several important secondary metabolites in the pharmaceutical industry.
Medicinal plants serve as a vital source for the development of health-promoting biomaterials because of their diverse bioactive compounds with therapeutic potential (Luo et al. 2024). Modern advancements in natural biomaterials have significantly enhanced health applications, for instance, the development of surface-functionalized blood-contacting biomaterials has shown great promise in preventing coagulation and promoting hemostasis (Wang et al. 2023). The species Crinum asiaticum, an Amaryllidaceae family member, is a perennial bulbous herbaceous plant that grows up to 2 m tall and has green leaves. It grows well in subtropical and tropical regions with fertile and moist soil and requires light shade to full sun (Mahomoodally et al. 2021). It is an important medicinal and ornamental plant that is abundantly found in mainland Japan, China, India, Sri Lanka, Myanmar, Thailand, and Malaysia (Rahman et al. 2012). In Ayurveda, it is referred to as naagadamani; however, in Malaysian and Papua New Guinean dialects, it is called Bakong, spider lily, Crinum lily, and poison bulb in English (Sura and Solleti 2020). In traditional medicine systems it has been used to treat earaches, vomiting, contusions, fractures, inflammatory joints, and sprains. Additionally, the plant has other important therapeutic effects, including anticancer, analgesic, antiviral, antimalarial, antibacterial, antifungal, and anti-inflammatory properties (Rakhi et al. 2024; Tan et al. 2019). Furthermore, this plant contains the following important phytoconstituents: Norgalanthamine, crinamine, flexinine, pratorimine, hamayne, stigmasterol, cycloartenol, cycloaudenol, ambeline, hippadine, ungeremine, criasbetaine, criasidine, and lycorine and its glucoside (Danquah et al. 2022). Globally, galanthamine (an isoquinoline alkaloid) is recommended as a medication for patients with dementia, schizophrenia, and Alzheimer’s diseases because it enhances cholinergic nicotinic neurotransmission in central and peripheral nervous systems by selectively inhibiting acetylcholine esterase (Priyadharshini et al. 2020; Syeed et al. 2024). Lycorine exhibits anticancerous and cytotoxic activities (Georgiev et al. 2020). Flexinine possesses antiviral and cytotoxic properties (Ka et al. 2021). Stigmasterol possesses antiosteoarthritis, anti-inflammatory, immunomodulatory, antiparasitic, and neuroprotective properties (Bakrim et al. 2022; Bansal et al. 2023).
Generally, propagation of C. asiaticum is achieved by means of seeds and bulbs. However, as a result of excessive utilization of valuable phytocompounds, poor seed germination, and poor viability rates, this plant is threatened in their natural habitats (Sanyal et al. 2023). Modern biotechnological techniques have superseded traditional breeding approaches for multiple plants because of the presence of several phytocompounds (Bansal et al. 2024a). Additionally, micropropagation is an alternative effective technique for quick multiplication and proliferation of plants, production of pathogen free stocks, genetically modifying clones, and obtaining therapeutically important bioactive substances within a shorter period of time without the use of field farming (Murthy et al. 2023). Among other techniques, callogenesis has emerged as a valuable tool to produce plant-derived medicinal compounds (PDMCs) at a commercial scale (Cardoso et al. 2019) because almost every plant portion has the capability to generate callus and can be monitored indefinitely with regular subculturing (Efferth 2019). The callus culture often exhibits a wide range of secondary metabolites that are not limited to any particular group of compounds (Benjamin et al. 2019).
Gas chromatography-mass spectrometry (GC-MS) techniques allow detection and validation of different phytocompounds such as alkaloids, phenols, saponins, tannins, alcohol, steroids, and glycosides based on their respective retention times and spectra (Ashwini and Rajanna 2022). Several investigations demonstrated the importance of a number of variables and their influences on in vitro development of callus cultures, such as the plant genotype, explant type, plant growth regulators (PGRs), and media composition of media (Fatima et al. 2024; Hazrati et al. 2022). Such factors usually cause an increase in reactive oxygen species (ROS) such as superoxide, hydrogen peroxide, and hydroxyl radical, which inhibit cell growth and differentiation, resulting in oxidative damage and subsequent cell death (Bibi et al. 2018; Ozden and Karaaslan 2011). To cope with harmful effects of such radicals, plants have in-built antioxidative defense systems such as the presence of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) to prevent cellular damages. Recent innovations in the extraction of secondary metabolites from medicinal plants have integrated advanced computational techniques to enhance efficiency. Wan et al. (2024) used an ant colony algorithm-enabled back propagation neural network combined with response surface methodology to optimize the ultrasonic extraction of alkaloids from safflower seeds, thus achieving improved antioxidant activity. The present investigation aimed to optimize a callus induction protocol from bulb scales of C. asiaticum. This study is first of its kind to identify the metabolites present tissues raised in vivo and in vitro using GC-MS techniques. Nonenzymatic antioxidant [2, 2-diphenyl-1-picrylhydrazyl (DPPH)] and antioxidant enzymatic assays (SOD and POD) were conducted to evaluate the antioxidative capacity of C. asiaticum tissues.
All of the solvents and chemicals used during this investigation were of analytical grade and purchased from Himedia (Mumbai, India), SRL (Mumbai, India), and Sigma-Aldrich (St. Louis, MO, USA). The chemicals and reagents included Murashige and Skoog (MS) medium (Murashige and Skoog 1962), PGRs, methanol (high-performance liquid chromatography grade), DPPH, guaiacol, hydrogen peroxide, ethylenediaminetetraacetic acid (EDTA), polyvinylpyrrolidone (PVP), Triton X-100, Nitro blue tetrazolium (NBT), riboflavin, methionine, and sodium carbonate.
In this study, healthy bulbs of C. asiaticum were collected from Herbal Garden, Jamia Hamdard, New Delhi, India, and they were identified by Dr. S. N. Khan (Taxonomist, Jamia Hamdard, India) (voucher specimen number: JH/BOT/DAC/2022/07). The inner scales of the bulbs were used as explants. The surface sterilization for callus induction of the Crinum bulb scales was performed according to the protocol reported by Bibi et al. (2018). First, the explants were washed under running tap water, rinsed with 70% (v/v) ethanol for 2 min, and then rinsed with distilled water thrice. Finally, the explants were treated with 0.1% (w/v) mercuric chloride solution for 2 min and then rinsed with distilled water thrice. The surface-sterilized explants were then cut into small segments (4–5 cm) and placed into glass test tubes (16 × 125 mm in size) containing 0.8% (w/v) agar-solidified MS medium with 3% (w/v) sucrose and various concentrations of PGRs with a pH adjusted to 5.8. Afterward, the cultures were kept in a growth chamber with an air temperature of 24 °C (±2 °C), 16-h photoperiod, and 30 µmol·m−2·s−1 radiation provided by cool white fluorescent light at a relative humidity of 50% to 55%.
Sterilized inner scales of bulbs were inoculated on MS supplemented with varying concentrations (0.25–1.0 mg/L) of auxins [2,4-dichlorophenoxyacetic acid (2,4-D) and α-naphthalene acetic acid (NAA)] alone or in combination with cytokinin [6-benzylamino purine (BAP)] to induce callus. The cultures were subcultured for callus proliferation after every 4-week period. Callus induction frequency (explants with callus formation), fresh biomass, and texture of callus were noted after 4 weeks of subculture.
The methanolic extracts of each sample (bulb scales) and bulb scale calli (2, 4-D–induced, 0.5 mg/L) were prepared according to protocol of Hussain et al. (2019). The samples were shade-dried at room temperature and ground into fine powder using a mortar and pestle. Approximately 1.0 g of fine powder of each sample was extracted in methanol solvent (10 mL) in a rotary shaker at 120 rpm for 2 d. Then, the dissolved mixtures were filtered and subsequently concentrated under reduced pressure in a rotary evaporator. Thereafter, the extracts were redissolved in methanol and subjected to a GC-MS analysis. The metabolite profiling of methanolic extract of each sample was conducted using GC-MS-QP-2010 (Shimadzu, Tokyo, Japan) with the following program settings: instrument calibration was performed by adjusting the pressure and carrier gas flow rate; helium was a carrier gas with a column flow of 1.21 mL/min; and the injection temperature was 260 °C. The separation column used for GC-MS was the Rxi-5Sil MS GC Capillary Column (30 m, 0.25 mm inner diameter, 0.25 µm degrees of freedom). The oven temperature was programmed from 80 °C (hold time, 2 min) to 280 °C (hold time, 18 min). The ion source and interface temperatures were set at 220 °C and 270 °C, respectively, with a solvent cut time of 4.50 min. The total GC-MS running time was 35 min for each sample. The phytocompounds were identified by comparing their respective retention indices and mass spectra with those already listed in the National Institute of Standards and Technology (NIST) library and GCMS solution software (version 4.45 SP 1) (Shimadzu Corp., Columbia, MD, USA).
The DPPH radical scavenging assay was performed following the protocol of Baliyan et al. (2022). Briefly, 0.4 mL of methanolic extract was mixed with 3.6 mL of DPPH (0.024 w/v). An equivalent amount of methanol was used as blank/control and mixed with 3.6 ml DPPH. Then, the samples were kept at room temperature for 90 min in total dark, and each sample was prepared in triplicate. The absorbance of samples was finally measured at a wavelength of 517 nm using an ultraviolet Visible spectrophotometer (BL-295; Biolinkk, Delhi, India). The antioxidant activity was calculated using the following formula:where AC is the absorbance of the control and AS is the absorbance of the sample.
Approximately 1.0 g of fresh samples (bulb scale and bulb scale callus) were finely homogenized with 10 mL of phosphate buffer (pH = approximately 7.0) and centrifuged at 12,000 rpm for 30 min at 4 °C. The supernatant was collected and used for the POD assay. The POD activity was checked using the methodology of Haida and Hakiman (2019), wherein the assay mixture contained 0.5 mL enzyme extracts, 1.0 mL of 10 mM potassium-phosphate buffer (pH = 7.0), 0.5 mL of 1% guaiacol solution, and 0.5 mL hydrogen peroxide solution (0.4%). The final volume was made by adding 2.5 mL of distilled water. Similarly, a standard group was also prepared, but it did not include the enzyme extract. Changes in the absorbance were measured at 470 nm for 30 min, and the enzymatic assay was calculated as A = ΕLC, where A is the absorbance, E is the extinction coefficient (6.39 mM−1·cm−1), L is the path length (1.0 cm), and C is the enzyme concentration [units (U)/g fresh weight (FW) of samples].
Fresh samples (1.0 g) were extracted with 10 mL of 0.5M phosphate buffer (pH = approximately 7.2) comprising 3 mM EDTA, 1.0% (w/v) PVP, and 0.2% (v/v) TritonX-100; finally, they were centrifuged at 12,000 rpm (4 °C) for 14 min. The supernatant was collected and subjected to the SOD assay. The reaction mixture (3 mL) contained 50 mM phosphate buffer (pH = approximately 7.8), 2.25 mM NBT solution, 3.0 mM EDTA, 10 µM riboflavin, 45 µM methionine, 1.0 M Na2CO3, and 10 µL of enzyme extract. As the control, a whole reaction mixture devoid of any enzyme extract was used. Thereafter, the mixture was incubated for 10 min at 25 °C under 15 W fluorescent lights, and the absorbance was measured at 560 nm against the control by using an ultraviolet VIS spectrophotometer. The amount of SOD required to cause a 50% reduction in NBT under test conditions was determined as one unit of SOD enzyme activity, and the enzymatic activity was expressed as U/mg FW.
All the in vitro culture experiments had a completely randomized design with six replicates per treatment, and every experiment was repeated thrice. The antioxidant assays were performed in triplicate, with each experiment repeated thrice. The resulting data were statistically analyzed by using an analysis of variance and SPSS software (version 25). Data were expressed as the mean ± standard error, and the significance of the mean differences was determined using Duncan’s multiple range test at P ≤ 0.05 (Duncan 1955) and the unpaired t test with SAS software (version 9.4; SAS Institute, Inc., Cary, NC, USA).
The influence of various PGRs such as 2,4-D (alone), NAA, and BAP (in combination) on induction of the callus from bulb scale explants was tested. The callus started to emerge from the basal portion of scales after 1 week of inoculation (Fig. 1A). The PGR-free MS served as a control in which no callus induction and growth were noted. The auxins 2,4-D and NAA are both crucial to developing the callus; for example, 2,4-D alone showed a significantly higher incidence of callusing than that used in NAA and BAP combinations (Fig. 1B, Table 1). The callus induction frequency and FW were best noticed on 0.5 mg/L 2,4-D–fortified MS medium (95.83% and 3.2 g, respectively) (Fig. 2A). By further increasing the 2,4-D concentration, the callus induction efficiency was slightly reduced. In contrast, the lowest callusing (37.5%) was observed on MS medium with 1.0 mg/L BAP + 0.5 mg/L NAA added, with 0.4 g FW (Fig. 2B). After 8 weeks, a similar trend in callus biomass growth was noticed in all treatments. The explants treated with combinations of BAP and NAA produced a white friable callus, whereas those treated with 2,4-D alone produced a cream compact callus. All the other tested concentrations and combinations showed a mild to moderate callus formation response.
Citation: HortScience 60, 8; 10.21273/HORTSCI18602-25
Citation: HortScience 60, 8; 10.21273/HORTSCI18602-25
The GC-MS analyses of methanolic extracts of bulb scales and bulb scales calli (8 weeks old, treated with 0.50 mg/L 2,4-D) of C. asiaticum revealed a total of 34 and 31 different phytocompounds, respectively (Table 2, Figs. 3 and 4). Among the bioactives exclusively identified in bulb scale extract were gamma-sitosterol (C29H50O), with the highest peak area of 7.64% and retention time (RT) of 27.425 min, followed by alpha-d-methylglucopyranoside (C7H14O6; 5.55%), vitamin E (C29H50O2; 3.26%), propylhexedrine (C10H21N; 2.33%), and 3′,5′-dimethoxyacetophenone (C10H12O3; 2.09%), as well as other phytoconstituents such as cholestane-3-yl thiocyanate (0.71%), 1,3,4-eugenol (0.49%), and neophytadiene (0.41%). However, certain phytocompounds were detected in methanolic extract of bulb scale callus only, which include pyranone (3.18%), benzenepropanenitrile (3.18%), 24-ethylcholesterol (3.15%), and ethyl hexopyranoside (2.60%). Furthermore, approximately 19 bioactive compounds (41.3% of total compounds detected) were common in both samples at varied levels. The chemical structures of certain important phytocompounds (present in both extracts) have been presented in Fig. 5. The bulb scale extract had a higher concentration of compounds when compared with callus extract. The contents of palmitic acid methyl ester (6.74%), linoleic acid (7.74%), and stearic acid (5.71%) were higher in bulb scale extract than in the callus tissue extract (4.34%, 5.56%, and 3.82%, respectively). Similarly, the amount of methyl elaidate was lower in callus extract (15.96%) than in the bulb scale sample (22.20%). Guanosine was one such compound with an almost eight-fold increase in callus extract (34.61%) as compared with bulb scale (4.24%). A similar difference was detected in the concentrations of squalene and 24-epicampesterol in bulb scale extract (3.09% and 2.65%, respectively) and in the callus sample (1.58% and 1.12%, respectively).
Citation: HortScience 60, 8; 10.21273/HORTSCI18602-25
Citation: HortScience 60, 8; 10.21273/HORTSCI18602-25
Citation: HortScience 60, 8; 10.21273/HORTSCI18602-25
The methanolic extracts of bulb scale and bulb scale callus of C. asiaticum were subjected to DPPH, POD, and SOD assays, and the results are summarized in Fig. 6. Overall, each antioxidant activity (DPPH, POD, and SOD) was slightly higher in callus tissue than in the bulb scale extract. For the DPPH assay, the callus extract exhibited higher scavenging activity (49.32%) than that of bulb scale extract (35.23%). Similarly, the highest POD activity was noticed in the callus sample (0.51 ± 0.01 U/g FW) compared with that in bulb scale (0.26 ± 0.02 U/g FW). A similar trend was also observed in SOD activity, whereby the callus extract showed a slight increment in SOD activity (1.06 ± 0.01 U/mg FW) as compared with the bulb scale extract, with SOD activity of 0.91 ± 0.02 U/mg FW.
Citation: HortScience 60, 8; 10.21273/HORTSCI18602-25
In this present study, a various responses in callus induction and callus morphology/texture were noted with different combinations and concentrations of PGRs. The function of PGRs in stimulating callus is important because these signaling elements control cell cycle enzymes, and exogenous PGR application alters other endogenous hormone levels, which causes plant cell wall dissipation and the development process (Qahtan et al. 2022). In our study, prolific callus induction was noted from bulb scale on MS medium supplied with 2, 4-D as compared with NAA and BAP combinations because the chemical structures of PGRs differ, and each has a distinct impact on metabolic responses (Al-Hamidi et al. 2023). Similarly, 2,4-D showed high efficacy for inducing callus in several investigated plants such as Rhodophiala pratensis (Trujillo-Chacón et al. 2020) and Saussurea costus (Khan et al. 2021), and these observations were in accordance with our findings. Additionally, 2,4-D at a lower concentration promotes callogenesis because its higher concentration may act as a herbicide, leading to browning and reduced viability of tissues.
One of the most popular techniques for producing a consistent and continuous supply of secondary metabolites is in vitro propagation (Espinosa-Leal et al. 2018). To identify and compare metabolic profiles of the mother plant and the callus tissue of C. asiaticum obtained in vitro, the GC-MS analysis was conducted. The analyses revealed a total of 19 phytocompounds present in both the samples, but they differed in their contents; however, some phytocompounds were exclusively present in two different samples. Callus extract showed the exclusive presence of phytocompounds like pyranone, benzenepropane nitrile, oleoyl chloride, and stigmast-5-en-3-ol. This differential presence of bioactives in each sample occurred because of their spatial and temporal regulation; in other words, secondary metabolites accumulate in specific cells, organs, and tissues at specific growth phases/conditions (Pérez-Mejía et al. 2024). Other factors like PGR exposure, number of subcultures, genotype, photoperiod, and humidity also play crucial roles in determining the metabolic profile of each sample. Thus, an in vitro callus culture can be considered a potent source when producing therapeutically significant phytocompounds for commercial purposes.
The bulb scale extract displayed the presence of a wide variety of compounds with high pharmacological values. One such compound is gamma-sitosterol, which is widely known to exhibit antidiabetic, anti-inflammatory, antioxidant, anticancerous, and antihyperlipidemic activities (Ambavade et al. 2014). Vitamin E has a role in cancer prevention and protecting against neurodegenerative diseases because of its antioxidant and anti-inflammatory effects (Szymańska et al. 2017). Eugenol demonstrated a broad spectrum of biological activity (i.e., antifungal, antibacterial, anti-inflammatory, anticancer, antioxidant, analgesic, and antiparasitic properties) (Abdou et al. 2021). Similarly, the callus-derived methanolic extract showed the presence of various phytocompounds such as stigmast-5-en-3-ol, 24-ethylcholesterol and olealdehyde. Stigmast-5-en-3-ol belongs to the phytosterol category and has antidiabetic, antiproliferative, and antioxidative effects (Bolar et al. 2016; Fernando et al. 2018; Sujatha et al. 2010). Zhang et al. (2021) reported various activities of cholesterol and its derivatives against diseases and found antimicrobial, anticancerous, antipsychotic, and anti-inflammatory properties. Several other important phytocompounds such as guanosine, methyl ester of palmitic acid, linoleic acid, stearic acid, arachidic acid, squalene, and 24-epicampesterol were detected in both the extracts with diverse pharmacological activities. Squalene is a triterpenoid with anti-inflammatory, antineoplastic, and antiatherosclerotic effects (Lou-Bonafonte et al. 2018). Linoleic acid acts as a protective agent against various health disorders like diabetes, cancer, obesity, and atherosclerosis (Yang et al. 2015). Pyranone and its derivatives have applications in leukemia treatment (Peroković et al. 2020). Benzenepropane nitrile is a volatile compound that prevents various cardiovascular and neurodegenerative disorders (Lasekan and Azeez 2014).
The cultivated cells and regenerated tissues frequently experience stress under in vitro systems, leading to the production of ROS that lower the cell survival rate (Malik et al. 2018). To combat ROS toxicity, plant cells have inherent enzymatic (such as SOD, POD, CAT, glutathione reductase, and ascorbate peroxidase) and nonenzymatic defensive mechanisms. In our study, the antioxidant activities (DPPH, POD, and SOD) of callus were high compared with those of bulb scale extract, thus revealing adverse cellular conditions during cultivation. Similar reports of increased antioxidant activity in the in vitro samples of other investigated plant species are available (Abdulhafiz et al. 2020; Bansal et al. 2024b; Mishra et al. 2021).
The present study described the phytocompounds’ profiles of bulb scale and bulb scale callus in C. asiaticum. The bulb scales of C. asiaticum produced a positive response when inducing callus when exposed to different PGR concentrations. The obtained calli were subject to metabolite profiling using the GC-MS technique. Callus derived in vitro revealed the presence of diverse phytocompounds in varied quantities, indicating a rich reservoir of medicinally important metabolites like terpenoids, sterols, fatty acids. Because GC-MS allows the separation of low-molecular-weight and volatile compounds, other chromatographic techniques like ultra-high-performance liquid chromatography coupled with mass spectrometry or liquid chromatography tandem mass spectrometry should also be used. Additionally, the antioxidant activities (DPPH, POD, and SOD) of tissues were higher in the in vitro samples as compared with the in vivo samples. Thus, the present investigation may contribute to in silico molecular docking and bioprospecting studies of advanced pharmacological applications such as drug discovery.
Callus induction and growth from bulb scale explants of C. asiaticum. (A) Callus initiation after 1 week of inoculation. (B) Callus proliferation after 3 weeks. (Bars = 0.5 cm.)
Callus biomass growth (in grams) of C. asiaticum on (A) 2,4-D alone and (B) α-naphthalene acetic acid (NAA) + 6-benzylamino purine (BAP) (in combination) in amended Murashige and Skoog medium. Each value represents the mean ± standard error of three repeated experiments. Mean values followed by the different superscripts in a same column are significantly different from each other according to Duncan’s multiple range test at P ≤ 0.05 level.
Gas chromatography-mass spectrometry chromatograms of methanolic extracts of (A) bulb scale and (B) bulb scale callus of C. asiaticum representing the retention times with the corresponding peaks. The X-axis represents the time of elution of each compound and the Y-axis represents the intensity of the signal measured by the ion detector.
Venn diagram representing the proportion of phytocompounds detected by gas chromatography-mass spectrometry in bulb scale and callus extracts of C. asiaticum.
Chemical structure of some important phytocompounds identified in both the extracts (in vivo and callus samples). Images were taken from the PubChem compound database, National Centre for Biotechnology Information.
Antioxidant activities (A) 2, 2-diphenyl-1-picrylhydrazyl (DPPH) activity, (B) peroxidase (POD) activity, and (C) superoxide dismutase (SOD) activity of bulb scale and bulb scale callus of C. asiaticum. Each value represents the mean ± standard error of three replicates. *Significant at P ≤ 0.05 according to Student’s unpaired t test.
Contributor Notes
This research work is funded by the Department of Biotechnology (DBT/2020/JH/1336), New Delhi, India, and ongoing research funding program (ORF-2025-375), King Saud University, Riyadh, Saudi Arabia.
The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.
The authors declare no conflicts of interest.
The first author is thankful to the Department of Biotechnology (DBT) for financial support given as a Senior Research Fellowship. The authors are also grateful to the laboratory facilities provided by the Department of Botany, Jamia Hamdard, New Delhi. The authors acknowledge the ongoing research funding program (ORF-2025-375), King Saud University, Riyadh, Saudi Arabia.
Callus induction and growth from bulb scale explants of C. asiaticum. (A) Callus initiation after 1 week of inoculation. (B) Callus proliferation after 3 weeks. (Bars = 0.5 cm.)
Callus biomass growth (in grams) of C. asiaticum on (A) 2,4-D alone and (B) α-naphthalene acetic acid (NAA) + 6-benzylamino purine (BAP) (in combination) in amended Murashige and Skoog medium. Each value represents the mean ± standard error of three repeated experiments. Mean values followed by the different superscripts in a same column are significantly different from each other according to Duncan’s multiple range test at P ≤ 0.05 level.
Gas chromatography-mass spectrometry chromatograms of methanolic extracts of (A) bulb scale and (B) bulb scale callus of C. asiaticum representing the retention times with the corresponding peaks. The X-axis represents the time of elution of each compound and the Y-axis represents the intensity of the signal measured by the ion detector.
Venn diagram representing the proportion of phytocompounds detected by gas chromatography-mass spectrometry in bulb scale and callus extracts of C. asiaticum.
Chemical structure of some important phytocompounds identified in both the extracts (in vivo and callus samples). Images were taken from the PubChem compound database, National Centre for Biotechnology Information.
Antioxidant activities (A) 2, 2-diphenyl-1-picrylhydrazyl (DPPH) activity, (B) peroxidase (POD) activity, and (C) superoxide dismutase (SOD) activity of bulb scale and bulb scale callus of C. asiaticum. Each value represents the mean ± standard error of three replicates. *Significant at P ≤ 0.05 according to Student’s unpaired t test.
